1. Field of the Invention
The invention relates generally to the field of identification of DNA sequences, genes or chromosomes.
Generation of DNA Probes
Human genomic DNA is a mixture of unique sequences and repetitive sequences that are present in multiple copies throughout the genome. In some applications, nucleic acid hybridization probes to detect repetitive sequences are desirable. These probes have shown utility in the fields of fetal cell diagnostics, oncology, and cytogenetics. In other applications it is desirable to generate hybridization probes that anneal only to unique sequences of interest on a chromosome. Preparation of unique sequence probes is confounded by the presence of numerous classes of repetitive sequences throughout the genome of the organism (Hood et al., Molecular Biology of Eucaryotic Cells (Benjamin/Cummings Publishing Company, Menlo Park, Calif. 1975). The presence of repetitive sequences in hybridization probes will reduce the specificity of the probes because portions of the probe will bind to other repetitive sequences found outside the sequence of interest. Thus, to ensure binding of hybridization probes to a specific sequence of interest, efforts must be made to ensure that repetitive sequences ill the probe do not anneal to the target DNA outside the sequence of interest.
Recent contributions have addressed this question by inhibiting hybridization of the repetitive sequences with the use of unlabeled blocking nucleic acids (U.S. Pat. Nos. 5,447,841 and 6,596,479). Use of blocking nucleic acids in hybridizations is expensive, does not completely prevent hybridization of the repetitive sequences, and can distort genomic hybridization patterns (Newkirk et al., “Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect,” Nucleic Acids Res. vol 33 (22):e191 (2005)). Thus, methods that prevent hybridization of repeat sequences without the use of blocking DNA are necessary for optimal hybridization.
One means to achieve this is to remove unwanted repeat segments from the hybridization probes prior to hybridization. Techniques involving the removal of highly repetitive sequences have been previously described. Absorbents, like hydroxyapatite, provide a means to remove highly repetitive sequences from extracted DNA. Hyroxyapatite chromatography fractionates DNA on tie basis of duplex re-association conditions, such as temperature, salt concentration, or other stringencies. This procedure is cumbersome and varies with different sequences. Repeat DNA can also be removed by hybridization to immobilized DNA (Brison et al., “General Methods for Cloning Amplified DNA by Differential Screening with Genomic Probes,” Molecular and Cellular Biology, Vol. 2, pp. 578-587 (1982)). In all of these procedures, the physical removal of the repetitive sequences will depend upon the strict optimization of conditions with inherent variations based upon the base composition of the DNA sequence.
Several other methods to remove repetitive sequences from hybridization probes have been described. One method involves using a cross-linking agent to cross-link repetitive sequences either to directly prevent hybridization of repetitive sequences or to prevent amplification of repeat sequences in a PCR reaction. (U.S. Pat. No. 6,406,850). Another method uses PCR assisted affinity chromatography to remove repeats from hybridization probes (U.S. Pat. No. 6,569,621). Both of these methods rely on the use of labeled DNA to remove repeat sequences which makes these processes complex and difficult to reproduce. Further, both methods are time consuming, requiring multiple rounds of repeat removal to produce functional probes, suitable for use in fluorescent in situ hybridization (FISH) or other hybridization reactions requiring high target specificity.
The use of duplex specific nucleases which preferentially cleave double stranded deoxyribonucleic acid molecules has been described for sequence variant detection applications such as single nucleotide polymorphisms (US 2005/0164216; U.S. Pat. No. 6,541,204). The ability of the enzyme to preferentially cleave perfectly matched nucleic acid duplex polynucleotides as compared to single stranded provides a means for removing non-target double stranded DNA from the sample mixture.
The ability of these nucleases to specifically digest the duplex form of polynucleotides was discovered in the instant invention to provide substantial benefit in manufacturing unique target specific probes that do not require blocking DNA, thus eliminating the costs and interfering affect of blocking DNA, and providing a means for rapid, efficient and cost effective production of high specificity probes.
Detection of specific sequences in a genome makes use of the fact that DNA consists of a helix of two DNA strands and that this double strand is most stable when these two strands are homologues. The DNA consists of a phosphate-sugar phosphate backbone and to every sugar one of four different nitrogenous bases, cytosine guanine thymine or adenine, might be present. Homologue strands pair every cytosine with a guanine and every thymine with an adenine. When a labeled homologue sequence is added to a genome and the DNA is made single stranded, these labeled sequences will hybridize, under the right circumstances, to the specific homologue sequence in tie genome. For this in situ hybridization, a number of probes are available for different detections purposes and applications.
Whole Chromosome/Paint Probes (WCP)
WCP incorporates labeled DNA material, homologous to a specific chromosome. The material is obtained by flow sorting of metaphase chromosomes or by laser dissection from a metaphase spread which is amplified by PCR or a related technique. After labeling and applying it to a properly prepared nucleus, it will stain the target chromosome. However, such labeled probes will in addition stain other non-target chromosomes because of structural or repetitive sequence elements that are shared among some or all chromosomes. Accordingly in order to stain only those sequences originating from the intended chromosome of interest, these common repetitive elements are usually inhibited by hybridization with blocking DNA or other methods that block or remove non-specific interactions.
Multiple chromosome paints are also applied to a single nucleus. WCP are labeled by different fluorochromes or with a combination of fluorochromes, providing no limit to the amount of WCP's applied in a single hybridization. WCP's axe mainly used for karyotyping and to study translocations of large fragments, regions and subregions of chromosomes which are best observed in a metaphase spread of a nucleus.
Centromere Probes
Centromere probes are targeted to a 171 bp sequence that occurs in repetitive order in every centromeric region of the human chromosomes. All chromosomes have a slightly different sequence and because of this all chromosomes are detected separately when the right hybridization stringency is used. Only two chromosome pairs, 13 with 21 and 14 with 22, share the same repeat and cannot be detected independently. Generally, centromeric probes are produced from plasmids containing an insert from one or a few copies of the 171 pb repeat. These probes are able to be hybridized without the addition of blocking DNA because the 171 bp sequences do not occur outside the regions of interest.
Telomere Probes
Human telomeres consist of an array of short repetitive sequences (i.e. TTAGGG). This is repeated several times in different amounts for every chromosome and individual test subject age. This repetitive sequence is used as a probe that will stain all chromosomes although not every chromosome will stain equally strong. To detect tie telomeric end of chromosomes, mostly a sub-telomeric bacterial artificial chromosome (BAC) clone is used. This BAC clone contains repetitive sequences which should be blocked or removed during or before hybridization.
Comparative Genomic Hybridization (CGH) Probes
CGH is a process that involves hybridizing a test genome to a reference genome. The reference genome may take the form of a metaphase chromosome spread from a healthy individual or may be array based using probe sequences that represent all or part of a genome. Microarray probes made using BAC clones contain repetitive sequences which must be blocked prior to hybridization. However; blocking has the potential to cause a deviation in the results when compared to repeat depleted probes (Knol and Rogan, Nucleic Acids Research, 2005, Vol. 33, No. 22). Further, the blocking step increases the cost of hybridization assays. If the probe sequences are depleted of repetitive sequences, the blocking step of the labeled genomic DNA is not necessary, resulting ill a reduction in the cost and removal of any variation.
Gene Specific Probes
Gene specific probes are designed to detect a region of the genome containing a target gene or group of genes. These probes are used to detect amplifications or deletions of specific genomic areas which correlated to the expression level of the specific gene of interest. The coding sequence of the gene(s) itself is not large enough to generate a detection signal for the probe that is visible using standard fluorescence microscope. Therefore such gene specific probes are not limited to just the coding gene sequences (exons) but also involve non-coding (introns), regulatory or other sequences around the gene. Because of the large sequences encompassed within even a gene specific probe design they often suffer from the undesirable inclusion of unwanted repetitive sequences. When such material is then either labeled and used in hybridizations or used in hybridizations and then labeled, the unwanted sequences must be blocked or removed from the probe to be able to detect the gene area specifically.
Microarray Probes
Similar to CGH, microarray probes are fixed to a carrier. In general, automated robotic techniques are used to spot cDNA-PCR products or synthetic oligonucleotides on a slide or similar fixed surface. Also, techniques exist to synthesize sequences directly on a slide (Affimetrix, Inc, Santa Clara). The slides are hybridized with labeled cDNA or RNA in combination with different labeled cDNA or RNA as controls.
Coupling Reporter Molecules to DNA Probes
DNA probes are visualized by coupled reporter molecules. These molecules need to be incorporated in or attached to the DNA probe. One method utilizes a reporter molecule, having nucleotides linked to enzymatic reactions. Examples include incorporation by nick translation or a random prime reaction. Further, an amine coupled nucleotide, built in this way, is subsequently coupled directly or indirectly to reporter molecules. Coupling is done by chemical labeling of the DNA. An example is the coupling of a reporter molecule linked to a platinum group which forms a coordinative bond to the N7 position of guanine as used in ULS labeling (Kreatech Diagnostics, Amsterdam) and described in U.S. Pat. Nos. 5,580,990; 5,714,327; 5,985,566; 6,133,038; 6,248,531; 6,338,943; 6,406,850; and 6,797,818. Reporter molecules can be radioactive isotope, non-isotopic labels, digoxygenin, enzymes, biotin, avidin, streptavidin, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent and photoluminescent, (including fluorescent and phosphorescent), dyes, haptens, and the like.
Sample Preparation
To be able to detect the labeled probes bound to interphase chromosomes, the nucleus should maintain morphology during and after the FISH procedures. Using fixation, cells or nuclei are attached to a solid layer such as a microscope slide. Fixation before during or after attachment to the solid layer, provide reference for identification. Depending on the type of cell or tissue, the nuclei have to be accessible for probe DNA, usually by pre-treating with proteolytic enzymes, heat, alcohols, denaturants, detergent solutions or a combination of treatments. Probe and nucleic DNA are made single stranded by heat or alkali treatment and then allowed to hybridize.
Use of DNA Probes
Microarrays
One common use of microarrays is to determine the RNA expression profile of a suspect tissue, tumor, or microbe. By analyzing the RNA expression profile, a prognosis for the treatment and survival of the patient is proposed. The prognostic value of RNA microarrays for clinical usage has yet to be determined. Another common use of microarrays are array based CGH. With this technique an entire genome can be screened for amplifications and/or deletions of chromosomal regions
Microscopy
Cytogenetic analysis in pre and post natal testing is used to determine whether or not a fetus has a cytogenetic abnormality in a cell population from the fetus. Samples are frequently obtained through aminocenthesis, conducted in pregnant women who are considered to have an increased risk for cytogenetic abnormalities. Accordingly, these cells are investigated for cytogenetic abnormalities. The same type of investigations are performed to confirm cytogenetic abnormalities or investigate suspect cytogenetic abnormalities in cell populations obtained after delivery.
Assessing Fetal Cells in Maternal Blood
During pregnancy, fetal cells may enter into the maternal blood with increases in the number of these fetal cells found with trauma, (pre)-ecclampsy and abnormal pregnancies. In routine assessments of fetomaternal hemmorrhages, the frequently used Kleihauer-Betke test is based on the detection of red blood cells expressing fetal hemoglobin. For detection of cytogenetic abnormalities, nucleated cells from maternal blood are needed. The frequency of these cells is considerably lower and are estimated to be in the range of 1-10 fetal cells per mL of maternal blood. Nucleated red blood cells, trophoblast cells and the presence of hematopoietic progenitors that are of fetal origin provide a target for isolation and probe hybridization in the detection of cytogenetic abnormalities early in the pregnancies. To date a reliable and reproducible method to identify and assess the cytogenetic composition of these cells is not available. One of the main problems with this analysis is the loss of fetal cells at various steps throughout the procedure, resulting in inconsistent or inconclusive information.
Oncology
FISH is used to detect various kinds of chromosomal aberrations like translocations, deletions, amplifications, inversions, and duplications. These aberrations are detected in all types of cells and tissue. In leukemia, cells are isolated from blood or bone marrow for subsequent FISH analysis. In bladder cancer, cells are isolated from urine. Cells from solid tumors are obtained by puncture or excision of the tumor itself. Also, cells that are released by solid tumors are isolated from the blood and analyzed by FISH. The latter gives the opportunity to monitor tumor treatment closely in order to detect a chromosomal change in the tumor. In some types of cancer, FISH provides a prognosis of tumor progression or predicts the efficacy of specific medication. Commercially, the most used FISH tests are the BCR-ABL translocation FISH in Chronic myelogenous leukemia and the her2/neu gene amplification FISH in breast cancer.
Disseminated Tumor Cells
Methods for the characterization of not only tumor cells, but also rare cells, or other biological entities from biological samples have been previously described (U.S. Pat. No. 6,365,362). This two stage method requires efficient enrichment to ensure acquisition of target cells while eliminating a substantial amount of debris and other interfering substances prior to analysis, allowing for cellular examination by imaging techniques. The method combines elements of immunomagnetic enrichment with multi-parameter flow cytometry, microscopy and immunuocytochemical analysis in a uniquely automated way. The combination method is used to enrich and enumerate epithelial cells in blood samples, thus providing a tool for measuring cancer.
The two stage method has applications in cancer prognosis and survival for patients with metastatic cancer (WO 04076643). Based on the presence of morphologically intact circulating cancer cells in blood, this method is able to correlate the presence of circulating cancer cells of metastatic breast cancer patients with time to disease progression and survival. More specifically, the presence of five (5) or more circulating tumor cells per 7.5 milliliters provides a predictive value at the first follow-up, thus providing an early prognostic indicator of patient survival.
The specificity of the assay described above increases with the number of cells detected and is not sufficient in cases were only few (generally less than 5 circulating tumor cells) are detected. One solution to this problem is to provide detailed genetic information about suspected cancer cells. Accordingly, a method that would incorporate enrichment of a blood sample with multi-parametric image cytometry and multi-parametric genetic analysis on an individual suspect cancer cell would provide a complete profile and confirmatory mechanism to significantly improve current procedures for patient screening, assessing recurrence of disease, or overall survival.
Fluorescent in situ hybridization (FISH) has been described as a single mode of analysis in rare cell detection after enrichment as described in WO 00/60119; Meng et al. PNAS 101 (25): 9393-9398 (2004); Fehm et al. Clin Can Res 8: 2073-2084 (2002) and incorporated by reference herein. After epithelial cell enrichment, captured cells are screened by known hybridization methods and imaged on a microscope slide. Because of inherent technical variations and a lack of satisfactory confirmation of the genetic information, the hybridization pattern alone does not provide a level of clinical confidence that would be necessary for sensitive analysis, as in assessing samples with less than 5 target cells. Further, this method for FISH analysis is difficult to automate.
Coupling hybridization-based methods with immunocytochemistry in the analysis of individual cells has been previously described (U.S. Pat. No. 6,524,798). Simultaneous phenotypic and genotypic assessment of individual cells requires that the phenotypic characteristics remain stable after in situ hybridization preparatory steps and are limited in the choice of detectable labels. Typically, conventional in situ hybridization assays require the following steps: (1) denaturation with heat or alkali; (2) an optional step to reduce nonspecific binding; (3) hybridization of one or more nucleic acid probes to the target nucleic acid sequence; (4) removal of nucleic acid fragments not bound; and (5) detection of the hybridized probes. The reagents used to complete one or more of these steps (i.e. methanol wash) will alter antigen recognition in subsequent immunocytochemistry, cause small shifts in the position of target cells or completely removes the target cells, which introduces the possibility of mischaracterization of suspect cells.
Probe sets and methods for multi-parametric FISH analysis has been described in lung cancer (US 20030087248). A 3 probe combination resulting in 95% sensitivity for detecting bladder cancer in patients has also been described, see U.S. Pat. Nos. 6,376,188; 6,174,681. These methods lack the specificity and sensitivity for assessing small numbers of target cells, and thus a confirmatory assessment for early detection of disease state. They also do not provide a means for convenient automation.
One aspect of the present invention provides a confirmatory assay in the analysis of rare circulating cells by combining phenotypic and genotypic multiparametic analysis of an individually isolated target cell, resulting in a clinically significant level of sensitivity and, therefore, assurance to the clinician of any quantitative information acquired. Relevant disease states are assessed using extremely small (1, 2, 3, or 4) numbers of circulating tumor cells (CTC's) and provide a confirmation for early disease detection.